KR20160050716A - Brevibacillus R2a23 and its use - Google Patents
Brevibacillus R2a23 and its use Download PDFInfo
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Abstract
본 발명은 후자리움(Fusarium) 속 진균, 아스퍼질러스(Aspergillus) 속 진균, 또는 페니실리움(Penicillium) 속 진균 등에 대한 생장 억제 및 후자리움(Fusarium) 속 진균에 대한 포자 발아 억제 활성을 가진 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P) 균주 및 이러한 균주를 이용하여 농산물 등에서 후자리움 속 진균의 생장 및 포자 발아를 억제하는 방법에 관한 것이다. 본 발명의 브레비바실러스 속 R2a23 (KCTC18311P), 이를 포함하는 생균제 또는 조성물은 후자리움 속, 아스퍼질러스 속, 또는 페니실리움 속 진균에 대한 생장 및 후자리움 속 진균에 대한 포자 발아 억제 효과가 현저하게 우수하다.The present invention breather with Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, or penny room Solarium (Penicillium) in growth inhibition and Fusarium (Fusarium) spore germination inhibitory activity on the in fungi for such fungi Brevibacillus genus R2a23 (KCTC18311P), and a method for inhibiting the growth and spore germination of Fusarium spp. In agricultural products using such a strain. The prophylactic or therapeutic composition containing the Brevibacillus genus R2a23 (KCTC18311P) of the present invention and the prophylactic agent or composition containing the same has a remarkable inhibitory effect on spore germination for growth and fungi of the genus Fusarium, Aspergillus or Penicillium It is excellent.
Description
본 발명은 신규 균주(strain) 및 이의 용도에 관한 것이며, 특히 후자리움(Fusarium) 속 진균, 아스퍼질러스(Aspergillus) 속 진균, 또는 페니실리움(Penicillium) 속 진균 등에 대한 생장 억제 및 후자리움(Fusarium) 속 진균에 대한 포자 발아 억제 활성을 가진 신규 균주 및 이러한 균주를 이용하여 농산물 등에서 후자리움 속 진균의 생장 및 포자 발아를 억제하는 방법에 관한 것이다.The present invention relates to novel strains (strain), and relates to their use, in particular Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, or penny room Solarium (Penicillium) in the growth of such fungal inhibition and Fusarium ( The present invention relates to a novel strain having a spore germination inhibiting activity against a fungus belonging to the genus Fusarium and to a method for inhibiting the growth and spore germination of a fungus belonging to the genus Fusarium using such a strain.
곰팡이독소(mycotoxin)는 작물의 생육·저장·유통과정에서 오염된 곰팡이에 의해 생성되는 2차대사산물(secondary metabolites)로서 사람이나 가축에 급성적, 만성적 중독증(mycotoxicoses)을 일으킨다(Marasas, 1984; Desjardins, 2006). 독소생성 곰팡이에 의한 작물병해와 독소오염 농산물·식품의 섭취로 인한 인축의 중독증 피해는 매년 전 세계적으로 꾸준히 발생되기 때문에 WHO, FAO 등과 선진국에서는 곰팡이독소를 농산물·식품 내 위험물질로 간주하여 오염허용치 설정 등을 통해 발생과 전파를 규제하고 있다.Mycotoxins are secondary metabolites produced by contaminated fungi during the growth, storage, and distribution of crops and cause acyclovir and chronic mycotoxicoses in humans and livestock (Marasas, 1984; Desjardins, 2006). Crop disease and toxin pollution caused by toxin-producing fungi The toxic damage caused by the ingestion of agricultural products and foodstuffs is constantly occurring worldwide every year. Therefore, WHO, FAO and other advanced countries regard fungal toxins as dangerous substances in agricultural products and foods, And regulates the generation and propagation through the setting.
현재 우리나라 뿐 아니라 전 세계적으로 심각한 문제를 일으키는 곰팡이독소는 아스퍼질러스/페니실리움 속 곰팡이와 후자리움 속 곰팡이가 각각 생성하는 독소들이 대표적이다. 하지만 이들 독소의 발생지역이 서로 다르다. 아스퍼질러스/페니실리움 속 곰팡이가 생성하는 곰팡이독소로는 아플라톡신(Aflatoxin), 오크라톡신(Ochratoxin), 패툴린(Patulin) 등이 대표적이며 이들 독소는 주로 고온 다습한 열대 또는 아열대 지방의 곡물에서 발생한다. 반면 후자리움 곰팡이독소는 우리나라와 같은 온대지방에서 생산되는 곡물에서 흔히 발생하기 때문에 국내 농식품 산업 및 국민보건위생의 관점에서 후자리움 곰팡이와 독소 오염의 중요성이 대두되고 있다(Lee et al., 2010; Lee et al,. 2011; Lee et al., 2012). 독소 생성 후자리움 곰팡이에 의한 식물병 피해의 관점에서 후자리움 속 곰팡이(완전세대: Gibberella 속)는 전 세계적으로 분포하며 벼, 보리, 밀, 옥수수 등 많은 작물의 생육 중에 붉은곰팡이병, 이삭마름병, 이삭·줄기썩음병, 뿌리썩음병 등의 병을 일으켜 작물의 수량감소와 품질의 저하를 가져올 뿐 아니라 곰팡이 독소를 생성하여 중독증을 일으킨다. 병 발생 당해 연도의 피해 뿐 만 아니라 환경조건에 따라 여러 해에 걸쳐 병이 되풀이 발생하는 경향 때문에 후자리움 병에 의한 피해의 심각성은 전 세계적으로 더 해지고 있다.Toxins that cause serious problems not only in Korea but also all over the world are toxins produced by Aspergillus / Penicillium fungi and Fusarium fungi respectively. However, these toxins occur in different regions. The fungal toxins produced by Aspergillus / Penicillium fungi are Aflatoxin, Ochratoxin and Patulin. These toxins are mainly found in high temperature and high humidity tropical or subtropical grains Occurs. However, since the latter fungi toxin is common in cereals produced in temperate regions such as Korea, the importance of Fusarium fungi and toxin contamination is emerging from the perspective of domestic agro-food industry and public health hygiene (Lee et al., 2010; Lee et al., 2011; Lee et al., 2012). Toxin Generation From the viewpoint of damaging plant diseases caused by Liarium fungi, the genus Gibberella ( genus Gibberella ) is distributed throughout the world and has a wide variety of fungi such as red fungus, acanthosis, It causes diseases such as ear rot, stem rot, and root rot disease, resulting in a decrease in yield and quality of crops, as well as toxicosis by producing fungal toxins. Disease outbreaks The severity of the damage caused by the latter disease is increasing worldwide, not only because of the year but also because of the tendency to recur for many years depending on environmental conditions.
후자리움 곰팡이독소는 아스퍼질러스/페니실리움 독소와 달리 작물의 생육 중에 발생하며 주요 후자리움 독소로는 트리코테센(Trichothecene)계와 푸모니신(Fumonisin)계가 주종을 이루고 있다. 트리코테센계 독소는 F. graminearum, F. culmorum, F. poae, F. crookwellense, F. sporotrichioides 등의 후자리움 종에 의해 각종 맥류, 옥수수 등에서 주로 생성되며, 사람과 가축에 피부독성, 소화기관 및 순환기관에서의 출혈, 설사, 구토, 신경장애, 사료섭취 거부 등을 일으킨다(Marasas, 1984; Desjardins, 2006). 우리나라의 경우, 데옥시니발레놀(Deoxynivalenol)과 니발레놀(Nivalenol) 독소가 대표적인 트리코테센계 곰팡이독소로서 벼, 보리, 옥수수 등에서 검출되고 있으며, 이들 독소는 주로 F. graminearum (완전세대: Gibberella zeae)에 의해 생성된다 (Desjardins, 2006). 푸모니신계 곰팡이독소는 Gibberella fujikuroi complex [F. verticillioides (=F. moniliforme, 완전세대: Gibberella moniliformis), F. fujikuroi (완전세대: G. fujikuroi), F. proliferatum (완전세대: G. intermedia)], F. nygami, F. oxysporum 등에 의해 주로 옥수수 등에서 생성되며, 말, 노새, 당나귀 등에서 뇌백질 연화증, 돼지에서 폐부종(pulmonary edema) 증후군, 실험동물 흰쥐에서 간암, 간독성 및 신장독성을 일으킨다. 또한 사람에서는 식도암 발생과 깊은 관계가 있는 것으로 추정된다(Marasas, 2001).Fusarium fungus toxin, unlike aspergillus / penicillium toxin, occurs during the growth of crops, and the major secondary toxins are trichothecene and fumonisin. Tricothecenotoxin is produced mainly by Fusarium species such as F. graminearum, F. culmorum, F. poae, F. crookwellense and F. sporotrichioides , and it is produced in human beings and livestock by skin toxicity, digestive organs, Bleeding in circulatory organs, diarrhea, vomiting, neurological disorders, refusal to feed (Marasas, 1984; Desjardins, 2006). In Korea, Deoxynivalenol and Nivalenol toxins are typical tricothecene fungi toxins which are detected in rice, barley and corn. These toxins are mainly F. graminearum (complete generation: Gibberella zeae ) (Desjardins, 2006). Fu monitor Territories mycotoxins are Gibberella fujikuroi complex [F. verticillioides (= F. moniliforme, fully three: Gibberella moniliformis), F. fujikuroi (full generation: G. fujikuroi), F. proliferatum (full generation: G. intermedia)] , F. nygami , F. oxysporum, and the like, which cause cerebral leukodystrophy in horses, mules, and donkeys, pulmonary edema syndrome in pigs, liver cancer, hepatotoxicity and renal toxicity in experimental animal rats. It is also estimated that there is a strong relationship between the incidence of esophageal cancer in humans (Marasas, 2001).
상기와 같이 우리나라에서는 독성 후자리움 곰팡이에 의해 벼와 옥수수 등에 병해가 꾸준히 발생할 뿐 아니라 후자리움 곰팡이독소에 오염된 이병식물의 섭취로 인한 인축의 중독증 피해 가능성이 높다.As described above, in Korea, the toxicity of the latter fungus causes the disease to be caused not only in rice and corn, but also in the toxicity caused by ingestion of the fungus poisonous plant.
이러한 피해를 최소화하기 위하여 곡류에 우점하는 후자리움 곰팡이가 생산하는 독소를 저감시키며 친환경적인 기술의 개발이 필요하나 아직까지 미흡한 실정이다.To minimize these damages, it is necessary to develop eco - friendly technology that reduces the toxin produced by fusarium fungi, which is dominant in cereals, but it is still not enough.
따라서 본 발명이 이루고자 하는 기술적 과제는 독소를 생성하는 유해 진균, 특히 후자리움(Fusarium) 속 진균, 아스퍼질러스(Aspergillus) 속 진균, 또는 페니실리움(Penicillium) 속 진균 등의 생장 및 포자의 발아를 효과적이고 환경 친화적인 방법으로 억제할 수 있는 신규 균주 및 이러한 균주를 이용해 농산물, 식품 등에서 유해 진균의 생장 및 포자의 발아를 억제하는 방법을 제공하는 것이다.Thus germination of growth and spore, such aspect is harmful fungi, in particular Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, or penny room Solarium (Penicillium) in fungi that produce toxins another object of the present invention In an effective and environmentally friendly manner, and a method for inhibiting the growth of harmful fungi and germination of spores in agricultural products and foods using such strains.
상기 기술적 과제를 달성하기 위하여, 본 발명은 독소를 생성하는 유해 진균, 특히 후자리움(Fusarium) 속 진균, 아스퍼질러스(Aspergillus) 속 진균, 또는 페니실리움(Penicillium) 속 진균 등의 생장을 효과적으로 억제하는 신규 균주인 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P)을 제공한다.In order to accomplish the above object, the present invention provides a method for effectively and efficiently cultivating harmful fungi producing toxins, particularly fungus of the genus Fusarium , genus Aspergillus , or genus Penicillium , Brevibacillus genus R2a23 (KCTC18311P), which is a novel strain that inhibits the growth of human embryonic stem cells.
본 발명자들은 곡류 재배 토양에서 300여 종의 미생물을 분리한 후 배지에서 후자리움 속 독성 진균 8종(후자리움 그래미니아룸(Fusarium graminearum) PH-1, 후자리움 그래미니아룸(Fusarium graminearum) H-11, 후자리움 그래미니아룸(Fusarium graminearum) H7-4, 후자리움 그래미니아룸(Fusarium graminearum) H7-11, 후자리움 아시아티쿰(Fusarium asiaticum) SCK04, 후자리움 아시아티쿰(Fusarium asiaticum) R308, 후자리움 프롤리페라툼(Fusarium proliferatum), 및 후자리움 버티실리오이드(Fusarium verticillioides) 등)과 대치배양한 결과, 후자리움 속 진균에 길항력을 가지는 균주를 선발하였으며(도 1), 선발된 R2a23 균주는 16S rRNA 유전자의 염기서열 분석을 통해 브레비바실러스(Brevibacillus) 속으로 동정되었다(도 2).The present inventors isolated 300 kinds of microorganisms in the cultivated soils and then cultivated 8 kinds of fusarium toxin fungi ( Fusarium graminearum PH-1, Fusarium graminearum H- 11, Fusarium graminearum H7-4, Fusarium graminearum H7-11, Fusarium asiaticum SCK04, Fusarium asiaticum R308, Fusarium graminearum , Fusarium proliferatum , and Fusarium verticillioides ). As a result, a strain having resistance to the fungus of the latter genus was selected (Fig. 1), and the selected R2a23 strain Was identified in Brevibacillus through sequencing of the 16S rRNA gene (Fig. 2).
본 발명에 따른 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P) 균주는 통상적인 브레비바실러스 속 균주의 배양법에 의해 대량으로 배양할 수 있다. 배양배지로는 탄소원, 질소원, 비타민 및 미네랄로 구성된 배지를 사용할 수 있다. 예를 들어, NB(nutrient broth), TSB(tryptic soy broth) 액체 배지, 또는 TSA(tryptic soy agar) 고체배지 등을 사용할 수 있다. 배양은 통상의 배양 조건으로 수행할 수 있으며, 예를 들어 약 37℃에서 약 10-72시간 동안 배양할 수 있다. 배양액 중의 배양배지를 제거하고 농축된 균체만을 회수하기 위해 원심분리 또는 여과과정을 거칠수 있으며 이러한 단계는 당업자의 필요에 따라 수행할 수 있다. 농축된 균체는 통상적인 방법에 따라 냉동하거나 냉동 건조하여 그 활성을 잃지 않도록 보존할 수 있다.The strain Brevibacillus strain R2a23 (KCTC18311P) according to the present invention can be cultured in a large amount by a conventional culture method of Brevibacillus sp. Strain. As the culture medium, a medium composed of carbon source, nitrogen source, vitamins and minerals can be used. For example, NB (nutrient broth), TSB (tryptic soy broth) liquid medium, or TSA (tryptic soy agar) solid medium can be used. The culture can be carried out under normal culture conditions, for example, at about 37 ° C for about 10-72 hours. The culture medium in the culture broth can be removed and centrifuged or filtered to recover only the concentrated cells, and these steps can be carried out according to the needs of a person skilled in the art. The concentrated microbial cells can be preserved by freezing or freeze-drying according to a conventional method so as not to lose their activity.
또한, 본 발명에 따른 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P)균주는 당업계에 공지된 통상적인 물리화학적 돌연 변이 방법 등에 의해 이와 동등한 활성을 가지거나 또는 이보다 우수한 활성을 가지도록 개선 또는 개량될 수 있다.
The Brevibacillus genus R2a23 (KCTC18311P) strain according to the present invention may be modified or improved to have an activity equivalent thereto by a conventional physicochemical mutation method or the like known in the art or to have an activity superior thereto .
본 발명은 또한 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P) 균주를 포함하는 항진균 활성을 갖는 생균제를 제공한다.The present invention also provides a probiotic having an antifungal activity comprising a strain of Brevibacillus genus R2a23 (KCTC18311P).
또한, 본 발명은 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P) 균주를 포함하는 유해 진균 억제용 조성물을 제공한다.In addition, the present invention provides a composition for suppressing harmful fungi, which comprises a strain of Brevibacillus genus R2a23 (KCTC18311P).
상기 생균제 또는 조성물에서 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P) 균주는 균주 그 자체에 한정되는 것이 아니라, R2a23 균주의 파쇄된 세포벽 분획, 생균, 사균, 건조균, 또는 이의 배양물 또는 배양액, 또는 이의 배양액 또는 배양물로부터 분리된 후자리움(Fusarium) 속, 아스퍼질러스(Aspergillus) 속, 페니실리움(Penicillium) 속, 알터나리아 종(Alternaria sp.), 에피코쿰 종(Epicoccum sp.), 또는 에피코쿰 니그룸(Epicoccum nigrum)에 대한 길항 물질일 수 있다. 또한, 상기 생균제 또는 조성물은 적합한 부형제 또는 담체를 추가로 포함할 수 있다. 상기 배양액은 본 발명에 따른 균주를 적합한 액체 배지에서 배양한 배양액 자체, 상기 배양액을 여과 또는 원심분리하여 균주를 제거한 여액(여과액 또는 원심분리한 상등액), 상기 배양액을 초음파 처리하거나 상기 배양액에 용해효소(lysozyme)를 처리하여 수득한 세포 파쇄액 등을 포함하나 이에 한정되는 것은 아니다. 또한 상기 생균제 또는 조성물은 고체배지 배양물, 또는 상기 배양액의 건조물 또는 추출물 등의 배양물을 함유할 수 있으나, 이에 한정되는 것은 아니다.
The strain Brevibacillus strain R2a23 (KCTC18311P) is not limited to the strain itself but may be a cell wall fraction, a live cell, a dead bacterium, a dried bacterium, or a culture or culture thereof of R2a23 strain, or Aspergillus spp., Penicillium spp . , Alternaria spp. , Epicoccum sp. Spp. , Escherichia spp . , Fusarium spp. Isolated from its culture or culture, Or an antagonist against Epicoccum nigrum . In addition, the probiotics or compositions may further comprise suitable excipients or carriers. The culture medium may be a culture medium itself obtained by culturing the strain according to the present invention in a suitable liquid medium, a filtrate (filtrate or centrifuged supernatant) obtained by removing the strain by filtration or centrifugation of the culture medium, ultrasonication of the culture medium or dissolution in the culture medium A cell lysate obtained by treating an enzyme (lysozyme), and the like. The prophylactic or therapeutic composition may contain, but is not limited to, a solid culture broth or a culture such as a dried or extract of the culture broth.
본 발명의 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P) 균주가 특이적으로 길항 효과를 보이는 유해 진균으로는 후자리움(Fusarium) 속 진균, 아스퍼질러스(Aspergillus) 속 진균, 페니실리움(Penicillium) 속 진균, 알터나리아 종(Alternaria sp.), 에피코쿰 종(Epicoccum sp.), 및 에피코쿰 니그룸(Epicoccum nigrum) 등으로 이루어진 군에서 선택된 어느 하나일 수 있다.Bracelet according to the present invention the non-Bacillus (Brevibacillus) in R2a23 (KCTC18311P) as a harmful fungi strains showing the antagonistic effects specifically to the Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, penny room Solarium (Penicillium) And may be any one selected from the group consisting of fungi, Alternaria sp. , Epicoccum sp. , Epicoccum nigrum , and the like.
상기 후자리움(Fusarium) 속 진균은 후자리움 그래미니아룸(Fusarium graminearum) PH-1, 후자리움 그래미니아룸(Fusarium graminearum) H-11, 후자리움 그래미니아룸(Fusarium graminearum) H7-4, 후자리움 그래미니아룸(Fusarium graminearum) H7-11, 후자리움 아시아티쿰(Fusarium asiaticum) SCK04, 후자리움 아시아티쿰(Fusarium asiaticum) R308, 후자리움 프롤리페라툼(Fusarium proliferatum), 후자리움 버티실리오이드(Fusarium verticillioides), 후자리움 옥시스포룸(Fusarium oxysporum), 후자리움 아르메니아쿰(Fusarium armeniacum), 후자리움 안토필룸(Fusarium anthophilum), 후자리움 아베나세움(Fusarium avenaceum), 후자리움 바빈다(Fusarium babinda), 후자리움 비고니에(Fusarium begoniae), 후자리움 서시나툼(Fusarium circinatum), 후자리움 콘센트리쿰(Fusarium concentricum), 후자리움 크룩웰렌스(Fusarium crookwellense), 후자리움 에퀴세티(Fusarium equiseti), 후자리움 후지쿠로이(Fusarium fujikuroi), 후자리움 글로보섬(Fusarium globosum), 후자리움 글라미눔/헤테로스포럼(Fusarium graminum/heterosporum), 후자리움 콘줌(Fusarium konzum), 후자리움 락티스(Fusarium lactis), 후자리움 랑세티에(Fusarium langsethiae), 후자리움 나피폼(Fusarium napiforme), 후자리움 니시카도이(Fusarium nisikadoi), 후자리움 니가마이(Fusarium nygamai), 후자리움 필로필룸(Fusarium phyllophilum), 후자리움 포에(Fusarium poae), 후자리움 슈도안토필룸(Fusarium pseudoanthophilum), 후자리움 슈도그래미니아룸(Fusarium pseudograminearum), 후자리움 라미게눔(Fusarium ramigenum), 후자리움 레돌렌스(Fusarium redolens), 후자리움 사카리(Fusarium sacchari), 후자리움 삼부시눔(Fusarium sambucinum), 후자리움 실피(Fusarium scirpi), 후자리움 스포로트리키오이드(Fusarium sporotrichioides), 후자리움 스테릴리히포섬(Fusarium sterilihyphosum), 후자리움 서브글루티난스(Fusarium subglutinans), 후자리움 숙시세(Fusarium succisae), 후자리움 탑시눔(Fusarium thapsinum), 후자리움 트리신툼(Fusarium tricinctum), 및 후자리움 베네나툼(Fusarium venenatum) 등이 있다.The fungi belonging to the genus Fusarium are Fusarium graminearum PH-1, Fusarium graminearum H-11, Fusarium graminearum H7-4, Fusarium graminearum , California Grammy Room (Fusarium graminearum) H7-11, Fusarium Asia tikum (Fusarium asiaticum) SCK04, Fusarium Asia tikum (Fusarium asiaticum) R308, Fusarium program Raleigh Blow Tomb (Fusarium proliferatum), dE (Fusarium Fusarium Bertie Sicily cucumber verticillioides , Fusarium oxysporum , Fusarium armeniacum , Fusarium anthophilum , Fusarium avenaceum , Fusarium babinda , Fusarium remarks to you (Fusarium begoniae), Fusarium stood natum (Fusarium circinatum), Fusarium outlet rikum (Fusarium concentricum), Fusarium Crooked wells Lawrence (Fusarium crookwellense), then Leeum ekwi Cassetti (Fusarium equiseti), Fusarium Fuji Kuroishi (Fusarium fujikuroi), Fusarium Globo Island (Fusarium globosum), Fusarium articles Lamy num / hetero's forum (Fusarium graminum / heterosporum), Fusarium konjum (Fusarium konzum Fusarium lactis , Fusarium langsethiae , Fusarium napiforme , Fusarium nisikadoi , Fusarium nygamai , and the latter, Fusarium species such as Fusarium phyllophilum , Fusarium poae , Fusarium pseudoanthophilum , Fusarium pseudograminearum , Fusarium ramigenum , Fusarium species such as Fusarium redolens , Fusarium sacchari , Fusarium sambucinum , Fusarium scirpi , Fiumarium sprotriquioide ( F usarium sporotrichioides), Fusarium Ste Lily Hippo Island (Fusarium sterilihyphosum), Fusarium sub-article Ruti nonce (Fusarium subglutinans), Fusarium Ladies ticker (Fusarium succisae), Fusarium tapsi num (Fusarium thapsinum), Fusarium tree sintum (Fusarium tricinctum , and Fusarium venenatum .
상기 아스퍼질러스(Aspergillus) 속 진균은 아스퍼질러스 암스텔로다미(Aspergillus amstelodami), 아스퍼질러스 푸미가투스(Aspergillus fumigatus), 아스퍼질러스 클레로티오룸(Aspergillus schlerotiorum), 및 아스퍼질러스 트리티시(Aspergillus tritici) 등이 있다.The Aspergillus (Aspergillus) in fungi Aspergillus cancer seutelro Damiani (Aspergillus amstelodami), Aspergillus Fu fumigatus (Aspergillus fumigatus), Aspergillus Klee as Tio Room (Aspergillus schlerotiorum), and Aspergillus Tree Tee And Aspergillus tritici .
상기 페니실리움(Penicillium) 속 진균은 페니실리움 아우란티오그리세움(Penicillium aurantiogriseum), 페니실리움 시트리눔(Penicillium citrinum), 및 페니실리움 비리디카툼(Penicillium viridicatum) 등이 있다.
The penny room Solarium (Penicillium) in fungi and the like penny room Solarium brother is thio draw-raised (Penicillium aurantiogriseum), Penny room Solarium sheet rinum (Penicillium citrinum), and Penny room Solarium irregularities dicarboxylic Tomb (Penicillium viridicatum).
또한, 본 발명은 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P) 균주로 처리하는 유해 진균을 억제하는 방법을 제공한다. 바람직하게는, 본 발명의 유해 진균을 억제하는 방법은 후자리움(Fusarium) 속, 아스퍼질러스(Aspergillus) 속, 페니실리움(Penicillium) 속, 알터나리아 종(Alternaria sp.), 에피코쿰 종(Epicoccum sp.), 또는 에피코쿰 니그룸(Epicoccum nigrum) 등의 생장 및 후자리움(Fusarium) 속 진균에 대한 포자 발아 억제에 효과적으로 사용될 수 있다. 상기 방법은 배양액 또는 배양물로부터 분리된 후자리움(Fusarium) 속, 아스퍼질러스(Aspergillus) 속, 페니실리움(Penicillium) 속, 알터나리아 종(Alternaria sp.), 에피코쿰 종(Epicoccum sp.), 및 에피코쿰 니그룸(Epicoccum nigrum) 등에 대한 길항 물질을 처리하는 방법을 포함할 수 있다.
The present invention also provides a method for inhibiting harmful fungi by treating with a strain of Brevibacillus genus R2a23 (KCTC18311P). Preferably, the method for inhibiting the noxious fungus of the present invention is a method for suppressing harmful fungi of the genus Fusarium , the genus Aspergillus , the genus Penicillium , the genus Alternaria sp. , Epicoccum sp. , Or Epicoccum nigrum , and for the suppression of spore germination against the fungi of the genus Fusarium . The method comprising culture medium or separated from the culture Fusarium (Fusarium) genus Aspergillus (Aspergillus), A penny room Solarium (Penicillium), An Alterna Ria species (Alternaria sp.), Epi kokum species (Epicoccum sp. ), And Epicoccum nigrum and the like.
본 발명에 따른 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P), 이의 배양액 또는 배양물, 또는 이의 배양액 또는 배양물로부터 분리된 길항물질을 포함하는 것을 특징으로 하는 유해균 억제용 조성물은 식품, 사료 첨가제, 또는 세제 첨가제 등으로 이용될 수 있다.The composition for inhibiting harmful bacteria according to the present invention, which comprises a culture or culture of Brevibacillus genus R2a23 (KCTC18311P), a culture or a culture thereof, or an antagonistic substance isolated from the culture or culture thereof, Or a detergent additive.
본 발명의 브레비바실러스(Brevibacillus) 속 R2a23 (KCTC18311P), 이를 포함하는 생균제 또는 조성물은 후자리움(Fusarium) 속 진균, 아스퍼질러스(Aspergillus) 속 진균, 또는 페니실리움(Penicillium) 속 진균 등에 대한 생장 억제 및 포자 발아 억제 효과가 현저하게 우수하다.Of the present invention breather ratio Bacillus (Brevibacillus) in R2a23 (KCTC18311P), probiotic or compositions containing them are Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, or penny room Solarium (Penicillium) in fungi of the like The inhibition of growth and the effect of inhibiting spore germination are remarkably excellent.
도 1은 후자리움(Fusarium) 속 진균 8종을 브레비바실러스(Brevibacillus) 속 R2a23 균주와 대치 배양할 때 후자리움 속 진균의 생장 저해 효과가 나타난 사진이다.
도 2는 R2a23 균주가 브레비바실러스 속임을 나타내는 계통도이다.
도 3은 R2a23 균주를 영양 배지에서 배양한 후 배양여액에 곰팡이 포자현탁액(104/ml)을 혼합(배양여액 900ul + 포자현탁액 100ul)하고 25℃에서 배양하여 포자의 발아여부를 10일째 관찰한 결과이다.Fig. 1 shows eight Fusarium spp. Fungi belonging to the genus Brevibacillus This is a photograph showing the growth inhibitory effect of the fungi of the genus Fusarium at the time of confluence with the R2a23 strain.
Fig. 2 is a flow diagram showing that the R2a23 strain is Brevibacillus.
FIG. 3 shows that the strain R2a23 was cultured in a nutrient medium and then mixed with a fungal spore suspension (10 4 / ml) (culture filtrate 900ul + spore suspension 100ul) and cultured at 25 ° C to observe germination of spores at 10 days Results.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.
Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.
[실시예 1] [Example 1] BrevibacillusBrevibacillus R2a23의 선별 Selection of R2a23
곡류 재배 토양에서 분리한 300여종의 미생물을 후자리움 속 진균 8종(후자리움 그래미니아룸(Fusarium graminearum) PH-1, 후자리움 그래미니아룸(Fusarium graminearum) H-11, 후자리움 그래미니아룸(Fusarium graminearum) H7-4, 후자리움 그래미니아룸(Fusarium graminearum) H7-11, 후자리움 아시아티쿰(Fusarium asiaticum) SCK04, 후자리움 아시아티쿰(Fusarium asiaticum) R308, 후자리움 프롤리페라툼(Fusarium proliferatum), 후자리움 버티실리오이드(Fusarium verticillioides))과 25℃에서 5일간 대치 배양하였다. 상기 후자리움 속 진균 가운데 후자리움 프롤리페라툼(Fusarium proliferatum) 및 후자리움 버티실리오이드(Fusarium verticillioides)는 순천향대학교에서 분양 받았으며, 나머지 진균은 국립농업과학원에서 보유(자체 분리 또는 오래전 분양)하고 있는 균주를 사용하였다. Fusarium graminearum PH-1, Fusarium graminearum H-11, Fusarium graminarium ( Fusarium graminearum ), Fusarium graminearum , Fusarium graminearum , Fusarium graminearum H7-4, Fusarium graminearum H7-11, Fusarium asiaticum SCK04, Fusarium asiaticum R308, Fusarium proliferatum , , Fusarium verticillioides ) at 25 ° C for 5 days. Fusarium proliferatum and Fusarium verticillioides were distributed at Soonchunhyang University and the remaining fungi were retained by the National Institute of Agricultural Science Were used.
실험 결과, 도 1에 나타난 바와 같이 후자리움 속 진균에 대한 생장 억제능을 가지는 균주를 선발하였다.As a result of the experiment, a strain having the ability to inhibit the growth of Fusarium spp. Was selected as shown in Fig.
후술하는 16s rDNA 염기서열에 기초한 분자계통분류학적 분석을 통해 해당 균주가 브레비바실러스(Brevibacillus) 속임을 확인하고 이 균주를 R2a23로 명명하였으며, 2014년 7월 17일자로 한국생명공학연구원(KCTC)에 기탁하여 기탁번호 KCTC18311P를 부여받았다.
Based on the 16s rDNA nucleotide sequence described below, the strain was identified as Brevibacillus and named as R2a23. On July 17, 2014, the Korea Research Institute of Bioscience and Biotechnology (KCTC) And received the deposit number KCTC18311P.
[실시예 2] 선별된 [Example 2] BrevibacillusBrevibacillus R2a23 균주의 동정 Identification of strain R2a23
선별된 R2a23 균주를 동정하기 위해 16S rRNA 염기서열분석을 하였으며, R2a23 균주의 유전적 계통도를 확인하기 위해 EzBioCloud (http://www.ezbiocloud.net/)를 이용하여 16S rRNA 염기서열을 비교 분석하였다.16S rRNA sequencing was performed to identify selected strains of R2a23, and 16S rRNA sequences were compared and analyzed using EzBioCloud (http://www.ezbiocloud.net/) to confirm the genetic sequence of R2a23 strains .
본 발명의 선별 균주인 R2a23 균주의 16S rRNA 염기서열은 도 2에 나타나는 바와 같이 브레비바실러스(Brevibacillus) 속과 상당한 상동성을 보여 R2a23 균주는 브레비바실러스(Brevibacillus) 속으로 동정되었다.
As shown in FIG. 2, the 16S rRNA nucleotide sequence of R2a23 strain, which is a selective strain of the present invention, showed considerable homology with the genus Brevibacillus , and the R2a23 strain was identified as Brevibacillus genus.
[실시예 3] [Example 3] BrevibacillusBrevibacillus R2a23 균주의 포자 발아 억제 활성 평가 Evaluation of spore germination inhibitory activity of strain R2a23
본 발명에 따른 Brevibacillus R2a23 균주가 후자리움(Fusarium) 속 진균의 포자에 대하여 발아 억제 활성이 있는지 여부를 조사하였다. R2a23 균주를 영양배지에서 3일간 배양한 후 배양여액만 걸러 후자리움 속 진균 포자 현탁액(104/ml)을 혼합(배양여액 900 ul + 포자현탁액 100 ul)하여 25℃에서 배양하며 포자의 발아여부를 관찰하였다. 관찰 결과, 도 3에 나타난 바와 같이 관찰 10일째 후자리움 속 진균 8종(후자리움 그래미니아룸(Fusarium graminearum) PH-1, 후자리움 그래미니아룸(Fusarium graminearum) H-11, 후자리움 그래미니아룸(Fusarium graminearum) H7-4, 후자리움 그래미니아룸(Fusarium graminearum) H7-11, 후자리움 아시아티쿰(Fusarium asiaticum) SCK04, 후자리움 아시아티쿰(Fusarium asiaticum) R308, 후자리움 프롤리페라툼(Fusarium proliferatum), 후자리움 버티실리오이드(Fusarium verticillioides)) 모두에서 포자 발아가 억제됨을 확인하였다. 이러한 포자 발아 억제 효과는 21일까지 지속되었다.
Brevibacillus R2a23 according to the present invention The strain was examined for the germination inhibitory activity against the spores of the fungi belonging to the genus Fusarium . The R2a23 strain was cultivated in a nutrient medium for 3 days, and then mixed with a suspension of a fungal spore suspension (10 4 / ml) (culture filtrate, 900 μl + spore suspension, 100 μl) Respectively. As a result of observation, as shown in Fig. 3, on the 10th day of observation, eight kinds of Fusarium spp. ( Fusarium graminearum PH-1, Fusarium graminearum H-11, Fusarium graminearum H7-4, Fusarium graminearum H7-11, Fusarium asiaticum SCK04, Fusarium asiaticum R308, Fusarium proliferatum ), And Fusarium verticillioides ), respectively. This spore germination inhibition effect lasted to 21 days.
[실시예 4] [Example 4] BrevibacillusBrevibacillus R2a23 균주의 생장 억제 활성 평가 Evaluation of growth inhibitory activity of strain R2a23
Brevibacillus R2a23 균주의 생장 억제 활성을 평가하기 위하여, 실시예 1에 나타낸 후자리움 속 진균 8종 외의 후자리움 속, 아스퍼질러스 속, 페니실리움 속, 알터나리아 종, 및 에피코쿰 종 등에 Brevibacillus R2a23 균주를 대치배양하여 생장 억제 활성을 평가하였다.In order to evaluate the growth inhibiting activity of the Brevibacillus R2a23 strain, Example 1 Fusarium genus fungi 8 species other than the Fusarium genus, Aspergillus genus, Penny room Solarium in, Alterna Ria species, and epi kokum species like Brevibacillus R2a23 shown in And the growth inhibitory activity was evaluated.
실험 결과, Brevibacillus R2a23 균주가 후자리움 속 진균인 후자리움 옥시스포룸(Fusarium oxysporum), 후자리움 아르메니아쿰(Fusarium armeniacum), 후자리움 안토필룸(Fusarium anthophilum), 후자리움 아베나세움(Fusarium avenaceum), 후자리움 바빈다(Fusarium babinda), 후자리움 비고니에(Fusarium begoniae), 후자리움 서시나툼(Fusarium circinatum), 후자리움 콘센트리쿰(Fusarium concentricum), 후자리움 크룩웰렌스(Fusarium crookwellense), 후자리움 에퀴세티(Fusarium equiseti), 후자리움 후지쿠로이(Fusarium fujikuroi), 후자리움 글로보섬(Fusarium globosum), 후자리움 글라미눔/헤테로스포럼(Fusarium graminum/heterosporum), 후자리움 콘줌(Fusarium konzum), 후자리움 락티스(Fusarium lactis), 후자리움 랑세티에(Fusarium langsethiae), 후자리움 나피폼(Fusarium napiforme), 후자리움 니시카도이(Fusarium nisikadoi), 후자리움 니가마이(Fusarium nygamai), 후자리움 필로필룸(Fusarium phyllophilum), 후자리움 포에(Fusarium poae), 후자리움 슈도안토필룸(Fusarium pseudoanthophilum), 후자리움 슈도그래미니아룸(Fusarium pseudograminearum), 후자리움 라미게눔(Fusarium ramigenum), 후자리움 레돌렌스(Fusarium redolens), 후자리움 사카리(Fusarium sacchari), 후자리움 삼부시눔(Fusarium sambucinum), 후자리움 실피(Fusarium scirpi), 후자리움 스포로트리키오이드(Fusarium sporotrichioides), 후자리움 스테릴리히포섬(Fusarium sterilihyphosum), 후자리움 서브글루티난스(Fusarium subglutinans), 후자리움 숙시세(Fusarium succisae), 후자리움 탑시눔(Fusarium thapsinum), 후자리움 트리신툼(Fusarium tricinctum), 및 후자리움 베네나툼(Fusarium venenatum)에서 생장 억제 활성을 나타내는 것을 확인하였다.As a result, Brevibacillus R2a23 Wherein the strain is selected from the group consisting of Fusarium oxysporum , Fusarium armeniacum , Fusarium anthophilum , Fusarium avenaceum , Fusarium albinum , Fusarium species , such as Fusarium babinda , Fusarium begoniae , Fusarium circinatum , Fusarium concentricum , Fusarium crookwellense , Fusarium equiseti , , Fusarium Fuji Kuroishi, Fusarium lactis (Fusarium fujikuroi), Fusarium Globo island (Fusarium globosum), Fusarium articles Lamy num / hetero's forum (Fusarium graminum / heterosporum), Fusarium konjum (Fusarium konzum) ( Fusarium lactis , Fusarium langsethiae , Fusarium napiforme , Fusarium nisikadoi , Fusarium nygamy, amai), Fusarium Philo pilrum (Fusarium phyllophilum), the Fusarium PO (Fusarium poae), Fusarium pseudo St pilrum (Fusarium pseudoanthophilum), Fusarium pseudo Graeme California room (Fusarium pseudograminearum), Fusarium lamina genum (Fusarium ramigenum) , Fusarium redolens , Fusarium sacchari , Fusarium sambucinum , Fusarium scirpi , Fusarium sporotrichioides , and the latter. Fusarium sterilihyphosum , Fusarium subglutinans , Fusarium succisae , Fusarium thapsinum , Fusarium tricinctum , and the latter. It was confirmed that Fusarium venenatum showed growth inhibitory activity.
또한, Brevibacillus R2a23 균주는 아스퍼질러스 암스텔로다미(Aspergillus amstelodami), 아스퍼질러스 푸미가투스(Aspergillus fumigatus), 아스퍼질러스 클레로티오룸(Aspergillus schlerotiorum), 아스퍼질러스 트리티시(Aspergillus tritici), 페니실리움 아우란티오그리세움(Penicillium aurantiogriseum), 페니실리움 시트리눔(Penicillium citrinum), 페니실리움 비리디카툼(Penicillium viridicatum), 알터나리아 종(Alternaria sp.), 에피코쿰 종(Epicoccum sp.), 및 에피코쿰 니그룸(Epicoccum nigrum)에 대해서도 생장 억제 활성을 나타내는 것을 확인하였다.
In addition, Brevibacillus R2a23 Strain Aspergillus cancer seutelro Damiani (Aspergillus amstelodami), Aspergillus Fu fumigatus (Aspergillus fumigatus), Aspergillus Klee as Tio Room (Aspergillus schlerotiorum), Aspergillus Tree Tea City (Aspergillus tritici), Penny Room Solarium Such as Penicillium aurantiogriseum , Penicillium citrinum , Penicillium viridicatum , Alternaria sp. , Epicoccum sp. , Pseudomonas spp. And Epicoccum nigrum ( Epicoccum nigrum ).
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| CN112358974A (en) * | 2020-12-09 | 2021-02-12 | 昆明理工大学 | Endophytic fungus epicoccum nigrum FZT214 and application thereof |
| CN119639597A (en) * | 2024-10-17 | 2025-03-18 | 宁夏医科大学 | Bacillus tekii and application thereof in antagonizing fusarium |
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| KR20060123133A (en) * | 2003-11-11 | 2006-12-01 | 히게따 쇼유 가부시키가이샤 | Novel Brevibacillus supercinens and protein production method using this microorganism as host |
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| KR20060123133A (en) * | 2003-11-11 | 2006-12-01 | 히게따 쇼유 가부시키가이샤 | Novel Brevibacillus supercinens and protein production method using this microorganism as host |
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| CN112358974A (en) * | 2020-12-09 | 2021-02-12 | 昆明理工大学 | Endophytic fungus epicoccum nigrum FZT214 and application thereof |
| CN112358974B (en) * | 2020-12-09 | 2022-06-10 | 昆明理工大学 | A plant endophytic fungus FZT214 and its application |
| CN119639597A (en) * | 2024-10-17 | 2025-03-18 | 宁夏医科大学 | Bacillus tekii and application thereof in antagonizing fusarium |
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